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Evolution of Flavivirus sfRNA

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Evolution of Flavivirus sfRNA

Abstract

Arthropod-borne flaviviruses (FVs), including human pathogens such as Yellow fever virus (YFW), Japanese encephalitis virus (JEV) and Dengue virus (DENV) are a growing global health treat. The current outbreak of Zika virus (ZIKV) in Central and South America is supposedly linked to an increasing number of congenital microcephaly and Guillain-Barré syndromes. FV are small, single stranded positive-sense RNA viruses of 10-12kb length with highly structured untranslated regions (UTRs). The latter are associated with regulation of the viral life cycle, inducing processes such as genome cir-cularization, viral replication, packaging, and modulating pathogenicity. Here, we present a computational approach for identification and characterization of conserved structural elements in the UTRs of mosquito-borne flaviviruses (MBFV).
Evolution of Flavivirus sfRNA
Roman Ochsenreiter1, Andrea Tanzer1, Ivo L. Hofacker1,2, Michael T. Wolnger1,3*
1Department of Theoretical Chemistry, University of Vienna, Währingerstraße 17, 1090 Wien, Austria
2Bioinformatics and Computational Biology Research Group, University of Vienna, Währingerstraße 17, 1090 Wien, Austria
3Center for Anatomy and Cell Biology, Medical Unversity of Vienna, Währingerstraße 13, 1090 Wien, Austria
[1] Villordo, S.M., Carballeda, J.M.,Filomatori, C.V., Garmarnik, A.V. (2016), RNA Structure Duplications and Flavivirus Host Adaptation. Trends Microbiol 24(4), 270-283.
[2] Tanzer,A., Stadler, P.S. (2004), Molecular evolution of a microRNA cluster. J. Mol. Biol. 339:2, 327-35.
[3] Wolnger, M.T., Svrcek-Seiler, A., Flamm, C., Hofacker, I.L., Stadler, P.S. (2004), Ecient Computation of RNA Folding Dynamics. J.Phys.A: Math.Gen. 37: 4731-4741
[4] Rauscher S., Flamm C., Mandl, C., Heinz, F., Stadler, P.F. (1997), Secondary structure of the 3’-noncoding region of avivirus genomes: comparative analysis of base pairing probabilities RNA, 3(7):779–791
[5] Lorenz, R., Bernhart, S.H., Höner zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P.F., Hofacker, I.L. (2011), ViennaRNA Package 2.0, Algorithms for Molecular Biology 6:1, 26
[6] Nawrocki, E.P., Eddy S.R., Infernal 1.1: 100-fold faster RNA homology searches (2013), Bioinformatics, 29:2933-2935
Contact: michael.wolfinger@univie.ac.at - http://www.tbi.univie.ac.at
1. Introduction
6. Acknowledgements
2. sfRNA / xrRNA 5. Methods
3. MBFV SL element classes
4. Case Study - Zika virus SL1
Arthropod-borne aviviruses (FVs), including
human pathogens such as Yellow fever virus
(YFW), Japanese encephalitis virus (JEV) and
Dengue virus (DENV) are a growing global health
treat. The current outbreak of Zika virus (ZIKV)
in Central and South America is supposedly
linked to an increasing number of congenital
microcephaly and Guillain-Bar syndromes. FV
are small, single stranded positive-sense RNA
viruses of 10-12kb length with highly structured
untranslated regions (UTRs). The latter are
associated with regulation of the viral life cycle,
inducing processes such as genome cir-
cularization, viral replication, packaging, and
modulating pathogenicity [1].
Here, we present a computational approach for
identication and characterization of conserved
structural elements in the UTRs of mosquito-
borne aviviruses (MBFV).
This work was partly funded by the Austrian Science
Fund FWF projects "RNA regulation of the transcrip-
tome" (F43), "mRNAs von Viren: Evolution und
Struktur-Funktionsbeziehungen" (FWF-I-1303) and the
Austrian/French project "RNA-Lands" (FWF-I-1804-
N28 and ANR-14-CE34-0011).
We have analysed the SL elements of MBFV with co-
variance models (CM) [6] and identied ve unique
families of SL structures. Initial structural alignments
of SL elements gave strong evidence for hetero-
geneity even within single virus families. Conse-
quently we built initial CMs for distinct SL1 and SL2
sequences and iteratively rened and extended the
models by searching against MBFV 3'UTRs. Our CM
classication revealed independent evolutionary
conservation of SL hairpins YFVG, JEVG and DENVG.
To conrm our ndings, we measured sequence
similarity between these SL elements using a z-score
approach [2], which has previously been applied in
evolutionary studies of miRNAs. The resulting gene
tree conrmed our CM analysis, and supported our
hypothesis of independent deletion and duplication
events in the evolution of MBFV SL elements.
ZIKV, member of the Spondveni virus family is closely
related to DENVG. We found strong evidence that SL
elements in ZIKV were subject to a genomic reor-
ganization, compared to other DENV viruses. Our
analysis relates ZIKV SL1 to the DSL2 group, whereas
ZIKV SL2 is more similar to DSL1.
A single compensatory U to C mutation in asian
lineages of ZIKV critically inuences the capacity of
the RNA to fold into functional conformations. The
less stable african lineage folds into an unfavourable
stem-loop structure (Fig. 3). Contrary, the asian
lineage folds predominantly into structures that are
capable of building the three-way helix junction. The
mutation results in thermodynamic stabilization of
the functional form, thus allowing for ecient stalling
of Xrn1 and possibly increased virulence.
Identication and cha-
racterization of SL ele-
ments was performed
by two fundamentally
independent methods:
The identication step
utilized CMs of known
SL elements. Exten-
ding classic Markov
models, CMs can be un-
derstood as a variant of
stochastic contetxt free
grammars that allow for
simultaneous identi-
cation of sequence and
structure given a known
element. The CM, as
provided in the infernal
toolsuite [6], also gives
P-values, thus quant-
ifying the credibility of a
match.
Alternatively, we recon-
structed the phylogenic
history of SL elements
via a z-scoring ap-
proach [2]. To identify
similarities between SL
elements of dierent fa-
milies, we computed the signicance of the
alignment score as follows
Where is the identity score of 2 se-
quences in the alignment, and are
mean and variance of identity scores, after
repeated shuing of both sequences.
A U
--
P3
P2
P1
C
A
5' 3'
C
Fig. 2 Top: Assignment of SL ele-
ments from dierent MBFV families to
SL variants characterized by our CMs.
The structures represent the consen-
sus [5] of the groups. Color coding
indicates basepair conservation. ZIKV,
part of SPOVG shows rearrangement
of SL elements relative to DENVG.
Left: Schematic drawing of a SL hair-
pin, showing characteristic features.
Fig. 1 Architecture of MBFV 3'UTRs. RNA elements attri-
buted to halting Xrn1 are shown in red and blue. SL1 and
SL2 correspond to previously described SLII and SLIV in
West nile virus (WNV).
JSL1 JSL2
JEVG
AROVG+KOKVG
C
A
G
G
C
CAGA
A
_
_
_
___C C U G
CCACCG
G
A
UGUU_
G
G
U
A
G
A
C
G
G
U
G
C
U
G
C
C
U
G
U
A
C
G
C
A
C
A
G
A
C
C
A
C
_
_
_
_
_
UAAGUG
CCACU
A
G
_
______G__
U
G
_
C
G
A
A
_
G
A
G
U
G
C
U
G
U
C
U
G
G
C
U
A
G
C
U
AA
U
C
A
G
G
C
CAGA
A
A
A
A
___C C U G
CCACCG
G
AAGUUG
A
G
U
A
G
A
C
G
G
U
G
C
U
G
C
C
U
G
C
A
C
A
G
A
C
C
A
C
A
A
A
A
____
_GUG
CCACUCU
G_C
G
G
A
G
A
G
U
G
C
A
G
U
C
U
G
_
AG
CG
CG
C
NTAVG
C
A
G
G
C
C
A
G
_
_
U
C
A
A
UGCCUG
CCACCG
G
AAGUUG
G
A
U
G
A
C
G
G
U
G
C
U
G
C
C
U
G
C
U
C
C
C
A
G
G
C
C
A
C
A
G
G
UUUUGUG
C
C
A
C
U
AGCAUGC
A
G
U
G
C
U
G
C
C
U
G
U
A
C
A
G
G
C
C
U
G
AA_AA
G
C
C
A
C
C
U
G
A
U__CC
G
G
U
G
A
A
G
G
U
G
C
U
G
C
C
U
G
U
A
C
A
G
A
U
C
_
C
G
AAA
G
G
C
C
A
C
C
A
G
U
A
U
GGUG
C
A
A
A
A
C
U
G
G
U
G
C
U
A
U
C
U
G
U
G
C
GA
U
CM
DSL1 DSL2
C
A
G
G
C
C
G
A
G
_
_
_
_
__AACG
C
CAUGGC
A
CGGA
A
G
A
A
G
C
C
A
U
G
C
U
G
C
C
U
G
CG
C
A
G
G
C
C
G
G
A
U
_
____UA
AGC
C
AUAGU
A
CGGA
A
A
A
A
A
C
U
A
U
G
C
U
G
C
C
U
G
C
A
G
G
C
C
U
G
C
U
A
G
UCAGCCACAGCUU
GGG
G
A
A
A
G
C
U
G
U
GC
A
G
C
C
U
G
GC
UA
C
A
G
G
C
C
G
A
G
A
ACGC
C
A
U
G
G
C
A
CGGAA
G
A
A
G
C
C
A
U
G
C
U
G
C
C
U
G
(SL-1)
(SL-2)
C
A
G
G
C
C
A
C
_
__AA
A
_
GC
C
ACGG
C
UU
GAG
C
A
A
A
C
C
G
U
G
C
U
G
C
C
U
G
U
A
A
U
C
A
G
G
C
C
U
G
C
U
A
G
UCAGCCACAGCUU
GGG
G
A
A
A
G
C
U
G
U
GC
A
G
C
C
U
G
GC
UA
CG
CG
GC
C
G
G
C
A
U
G
C
A
U
DENVG
SPOVG CM
YSL
YFG
C
A
G
C
C
C
A
U
C
A
U
_
_
_
_
_
___A
AUGAUG
CCAUGGCU
AAGCU
G
U
G
A
G
G
C
C
A
U
G
C
U
G
G
C
U
G
A
U
C
A
G
_
_
_
_
____C
C
C
A
U
C
A
U
AA
UGAUGCCAUGGCU
AAGCU
G
U
G
A
G
G
C
C
A
U
G
C
U
G
G
C
U
G
A
U
U
A
CM
Upon FV infection, accumula-
tion of stable long non-coding
viral RNAs, termed subgeno-
mic aviviral RNAs (sfRNAs),
which dysregulate cellular
function with the aim of pro-
moting viral infections is ob-
served. Many MBFV produce
sfRNAs by eciently hi-
jacking the host cell's mRNA
degradation pathway. Mecha-
nistically, sfRNAs are gene-
rated by stalling the 5’-3’
exoribonuclease Xrn1 at cer-
tain structural elements in the
3’UTR of MBFV, termed xrRNA
(Xrn1-resistant RNA ele-
ments). These RNA structures
stall Xrn1, thereby providing
quantitative protection of
downstream RNA against de-
gradation.
MBFV typically have more
than one xrRNA element, each
with dierent capacity of stalling Xrn1,
thus enabling production of sfRNAs of
dierent lengths. Conserved RNA structu-
ral elements in viral 3’UTRs have been identied
in our group [4], some of which have later been
attributed to xrRNA functionality, specically
stem-loop (SL) and dumbbell (DB) elements.
C
A
G
G
C
C
U
G
C
U
A
G
U
C
A
GC
C
A
C
A
GUU
U
G
G
G
GA
A
A
G
C
U
G
U
G
C
A
G
C
C
U
G
open chain
E = - 15.7 kcal/mol
C
A
G
G
C
C
U
G
C
U
A
G
U
C
A
GC
C
A
C
AGUU
U
G
G
GG
A
A
A
G
C
U
G
U
G
C
A
G
C
C
U
G
E = -15.4kcal/mol
CAGG
CCUGCUAGU
CAGCCA
CAG
U
U
U
G
G
G
G
A
A
A
G
C
U
G
U
G
C
A
G
C
C
U
G
E = - 15.3 kcal/mol
C
A
G
G
C
C
U
G
C
U
A
G
U
CAGCC
ACAGUUU
GGG
G
A
A
A
G
C
U
G
U
G
C
A
G
C
C
U
G
E = -15.6 kcal/mol
10-2 100102104106
time
0
1
population probability
ZIKV SL1
african lineage
10-2 100102104106
time
0
1
population probability
10-2 100102104106
0
ZIKV SL1
asian lineag e
C
A
G
G
C
C
U
G
C
U
A
G
U
CAGCC
ACAGCUU
GGG
G
A
A
A
G
C
U
G
U
G
C
A
G
C
C
U
G
C
A
G
G
C
C
U
G
C
U
A
G
UCAGCCACAGCUU
GGG
G
A
A
A
G
C
U
G
U
GC
A
G
C
C
U
G
E = - 18.0 kcal/mol
E = -17.7 kcal/mol
open chain
Fig. 3 Reduced folding kinetics based on numerical in-
tegration of a Markov process on the coarse grained
energy landscapes [3] of african and asian lineage ZIKV
SL1. Simulations are run from the open chain confor-
mation to thermodynamic equilibrium. Each line cor-
responds to the temporal population of a macrostate,
characterized by its minimum structure. The compen-
satory U to C mutation is highlighted in green.
SL1 SL2 DB1 DB2 3'SL
Domain I Domain II Domain III
5'3'
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